Nextseq sequencing platform

ScRNA-seq with ex vivo sorted CD8⁺pentamer⁺ T cells was performed using SmartSeq2 (ref. ^{35 (link)}) with the following modifications: reverse-transcription (RT) and PCR amplification were performed as described^{35 (link)} with the exception of using ISPCR primer with biotin tagged at the 5′ end and increasing the number of cycles to 25. Sequencing libraries were prepared using the Nextera XT Library Preparation Kit (Illumina) and sequencing was performed on Illumina NextSeq sequencing platform with NextSeq Control Software v.4.

Genomic DNA was extracted using a DNeasy UltraClean DNA isolation kit according to the manufacturer’s instructions (Qiagen, Hilden, Germany). DNA was quantified using a Qubit^® fluorometer (Invitrogen, Carlsbad, CA, USA) high-sensitivity assay, before dilution to the required concentration in RNase-free water and purification on AMPure XP beads (Beckman Coulter, Brea, CA, USA). Sequencing libraries were prepared from 0.5 ng/µl of RNA free genomic DNA. A total of 282 isolates were included for genomic sequencing using the Nextera-XT DNA sample preparation kit (Illumina, San Diego, CA, USA) and whole-genome sequencing performed using the Illumina NextSeq sequencing platform, generating paired-end reads (2 × 150 bp).
Paired-end reads were quality-assessed and trimmed using FastQC and Trimmomatic as described above. Trimmed reads were assembled into contigs using SPAdes version 3.13.1 (Bankevich et al., 2012 (link)). Contigs shorter than 500 bp were discarded from analysis. Genome contamination and completeness was assessed using CheckM version 1.0.13. To confirm assembly quality, only genomes conforming to all the following criteria were included in further analysis: (i) contig N50 of >20 kbp (ii) 90% of assembled bases at >5× read coverage (iii) completeness of >95% (iv) contamination of <5% (v) complete 16S rRNA gene sequence.